Based on the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database and the population data from the Bureau of the Census, in 2000, 1.22 million new cases of invasive cancer (619,700 men, 600,400 women) were diagnosed and 552,200 people died from cancer in the United States. See Braunwald et al., HARRISON'S PRINCIPLES OF INTERNAL MEDICINE, 15th ed., McGraw-Hill (2001), at pp 491. Cancer is still the second leading cause of death behind heart disease in the United States. Id. Much resource and effort are invested in seeking effective ways to prevent and treat cancer.
Small Intestine Tumors
As pervasive as cancer is, tumors of the small intestine are a rare event (see, e.g., Juckett & Rosenberg, (1988) Mech. Ageing Dev. 43: 239-257). Over the years, preliminary experiments have reported attempts to determine the cause of this natural tumor-free environment. However, the results were inconclusive and the work was not pursued vigorously.
The small intestine's profound resistance to cancer can be demonstrated by the low number of small intestine cancer deaths compared to other gastrointestinal cancers. The fractional ratio of small intestine cancers compared to all others is only 0.0071. Parkin et al. eds (1997) Cancer Incidence in Five Continents, Vol. V (IARC Scientific Publications No. 143) Lyon, IARC. This is approximately the same for all the years examined and for all the various cohorts. Id. Incidence of small intestine tumors also follows the same trend as mortality (Parkin et al., eds., (1997), Cancer Incidence in Five Continents, Vol. VII (IARC Scientific Publications No. 143) Lyon, IARC). The low incidence rate is almost universally true for the human species, for all genetic pools, and over long periods of time. It also appears to be true for almost all other mammalian species (with the possible exception of two varieties of sheep). See Lingeman et al., (1972), J. of the National Cancer Institute 48: 325-346.
Additional observations, regarding the unusual nature of the small intestine, come from studies on radiation-induced illness (primarily from Nagasaki and Hiroshima) and in genetic diseases of the intestine. Large doses of high-energy radiation cause death by the destruction of the cells of the bone marrow and the epithelial cells lining the small intestine, both of which are easily damaged or destroyed by radiation. In bomb survivors that received a non-lethal dose of radiation, there is a much higher than normal incidence of cancers of the bone marrow at later times, but there is no increased incidence of cancers of the small intestine. See Shimizu et al. (1991), J. Radiat. Res. Supplement, 212-230.
In Familial Polyposis Coli, a genetic disease, it is known that patients have a predisposition to large intestine cancers. However, the potential for cancers of the small intestine is very low even in those individuals where the polyposis appears in the entire gastrointestinal tract. See Lightdale et al., (1982), Small Intestine, in: Cancer epidemiology and Prevention (eds, Schottenfield et al.,) W.B Saunders Co.
Finally, and most significantly, the spread of metastases from cancers arising in other sites to the small intestine is very rare. This suggests that whatever mechanism protecting the small intestine from local cancers also operates to block the occurrence of most other cancers as well.
The very low incidence of cancers in, or of, the small intestine is a robust fact of nature, and cries for explanation. The facts described above have been known for a long time. They have astonished many scientists and stimulated them to speculate on its causes. The low incidence of small intestine tumors cannot be attributed to the relative size of the organs of the digestive tract, since in humans the small intestine constitutes 90% of the length of the tract, and 98% of the absorptive surface area (Gabos et al., (1993) International J. of Epidemiology 22: 198-206). This is also true for other animals as well. The low incidence cannot be attributed to a low cell turnover rate, as it is in neural cells and muscle cells, since the epithelium of the small intestine crypts have the fastest turnover rates of all organs of the body. Potten and Loeffler (1990, Development 110: 1001-1020) have commented, “Since there are about 7.5×105 crypts in the ileum of the mouse there must be between 3×106 and 1.2×107 stem cells in the ileum. Thus, there are between 109 and 1010 stem cell divisions in the mouse small intestine in its 3-year life span. With a spontaneous mutation rate of approximately 1 in 106, about 103 or 104 spontaneous mutations would be expected, i.e. about 1-10 per day. It is therefore surprising that the small intestine rarely develops cancer. It must be very well protected by ill-understood mechanisms.”
Ashley and Wells (1988, Seminars in Oncology 15: 116-128) listed various speculations regarding the protected status of the small intestine. They include: a) protection from exposure to noxious substances by the liquidity and rapid transit of its contents; b) the alkalinity of the interior lumen of the small intestine; c) the high concentrations of secretory immuno-globulins; d) the low bacterial concentration; and e) the presence of hydroxylases which may inactivate potential carcinogens. Another mechanism was proposed by Cairns (1975, Nature 255: 197-200). This involves the physical movement of epithelial cells, which begin as stem cells at the bottom of the crypt, move along the surface of the crypt and then the villus, and are finally shed into the lumen. This might prevent the accumulation of variant (abnormal) stem cells which may eventually be further mutated into transformed cells (cancer). None of these speculations have been proven.
In a paper by Bennett et al. (1951, Proceedings of Society for Experimental Biology 78: 790-791, referred as “Bennett” herein after), it was suggested that the small intestine contained a chemical inhibitory to tumor growth. Cells of the small intestine of mice were extracted and separated into 3 fractions—the pellet arising from a low speed centrifugation of the raw material (fraction 4), a pellet arising from a high speed centrifugation (fraction 90), and its supernatant (supernatant). Lymphosarcoma cells were incubated with each fraction for a specified time and both were inoculated into mice. The results were that few animals developed tumors using fraction 90, but a larger number of animals developed a tumor and died using the cells incubated with the supernatant and fraction 4. Similar extracts from liver, spleen and muscles failed to show any tumor inhibition. Bennett concluded that the tests confirmed their hypothesis. Bennett also showed in further tests that the supernatant activity was due to contamination with components of fraction 90 (containing microsomes), and could be eliminated by more careful preparation. Thus the supernatant was not active by itself. Bennett also stated that they have eliminated proteolytic enzymes as the probable cause of the activity.
In the mid 1970's, another group examined small intestine extracts (Chan et al., 1975, J. of the British Society of Gastroenterology 16: 50-52, referred as “Chan” herein after) and came to a different explanation of Bennett's result. Chan concluded that the in vitro incubation of tumor cells with small intestine extracts resulted in killing the cells prior to inoculation into animals; that the extracts kill normal cells as well as tumor cells; and that the activity was predominantly in the microsomal fraction and not in the supernatant. They suggested that the enzymatic activity of the epithelium was responsible for the observations.
In a paper by Calman (1974, Gut 15: 552-554), the anti-tumor effect in the small intestine was shown to require the presence of an intact immune system. Thymectomy and radiation was shown to allow tumors to be transplanted to the small intestine. In animals that received the same treatment, but with their immune system reconstituted with thymus grafts, tumors would again no longer grow in the small intestine. In both groups, tumors could be transplanted successfully to the stomach. Thus, the protective effect of the small intestine appeared to be related to an enhancement of the local immune system. However, the search for factor(s) that controlled the local immune system was not performed.
Protozoa and Immune Responses of their Hosts
Parasites have evolved multiple evasion strategies allowing them to survive in their primary host and to complete their life cycle through a series of stages that usually involve passage through the environment and often through secondary hosts. These strategies include antigen shedding, antigen shifting, living intracellularly for long periods, manipulating host cell biochemistry, infecting multiple organs, and encystment (see, e.g., Beverley, (1996), Cell 87: 787-789; Zambrano-Villa et al., (2002), Trends in Parasitol. 18: 272-278). Such mechanisms have been fine-tuned through evolutionary pressures to help the protozoan avoid the immune response of the host. In many cases, protozoans are so specialized in these avoidance mechanisms that they can only complete their life cycle in one host species or genera.
Apicomplexa are spore-forming single-celled parasites of animals and include species that cause various disease, such as malaria, coccidiosis, redwater disease, corridor disease, east coast fever and binary fever. Four major classes within the phylum Apicomplexa are: Coccidia (genera include, e.g., Besnoitia, Caryospora, Cryptosporidium, Eimeria, Frenkelia, Hammondia, Hepatozoon, Isospora, Lankesterella, Neospora, Sarcocystis, and Toxoplasma); Gregarinia (genera include, e.g., Gregarina, Monocystis, Ophriocystis, and Pseudomonocystis); Haemosporida or haemosporidians (genera include, e.g., Haemoproteus, Hepatocystis, and Plasmodium); and Piroplasmida or Piroplasmids (genera include, e.g., Babesia, Cytauxzoon, and Theileria). Seven species that infect humans have been identified (Plasmodium, Babesia, Cryptosporidium, Isospora, Cyclospora, Sarcocystis, Toxoplasma).
A defining characteristic of the Apicomplexa is a group of organelles found at one end—called the apical end—of the organism. This “apical complex” includes secretory organelles known as micronemes and rhoptries, polar rings composed of microtubules, and in some species a conoid which lies within the polar rings. At some point during their life cycle, members of the Apicomplexa either invade or attach to host cells. The apical organelles play a role in these host-parasite interactions.
A few Eimeria species are known to cause severe morbidity and occasionally mortality in certain animals. In particular, E. tenella, E. acervulina, and E. maxima are a problem in the chicken industry. The stress and crowded conditions exacerbate Eimeria infections and cause massive diarrhea and death. A similar problem can occur in the cattle industry, where crowding among calves can lead to severe disease. Usually, adult animals are immune to Eimeria but a low, asymptomatic infection is endemic in most vertebrates. Upon first exposure, a brief humoral immune response has been documented in animals (Faber et al., (2002), Vet. Parasitol. 104: 1-17; Allen & Fetterer, (2002), Clin. Microbiol. Rev. 15: 58-65; Rose, (1987), Eimeria, Isopora, and Cryptosporidium, in: Immune Responses in Parasitic Infections: Immunology, Immunopathology, and Immuneoprophylaxis (ed. E. J. L. Soulsby) pp 275-230), but this is replaced by a localized cellular immune response that appears to provide lasting protection throughout a host's life. The exact mechanism of this protection is not fully known and why low levels of the organism survive without undergoing the explosive growth of its life cycle remains a mystery.
Aliberti et al. (2003, Nature Immunology 4(5): 485-490) showed a purified protein, C-18, which is an isoform of Toxoplasma gondii cyclophilin (which does not contain a profilin domain), can stimulate production of interleukin-12 (IL-112) from dendritic cells (DCs). However, recombinant T. gondii C-18 showed reduced IL-12 stimulatory activity relative to the crude T. gondii tachyzoite extract (STAg).
The 19 kD Sporozoite Antigen
Vaccination against Eimeria has been the subject of substantial research in the animal husbandry of poultry, and to a lesser extent, cattle. Chemical prophylaxes are standard treatment in the poultry industry, but resistant strains of Eimeria are beginning to be a problem. The development of vaccines is considered an alternative approach and has focused on the use of surface antigens from the sporozoite stage, which is the stage that invades the epithelial layer of the small intestine (see e.g., Allen & Fetterer, (2002), Clin. Microbiol. Rev. 15: 58-65; Blackman & Bannister, (2001), Mol. Biochem. Parasitol. 117: 11-25; Min et al., (2001), Vaccine 20: 267-274). Merozoite surface antigens are also of interest because this protozoan stage is released after the first round of multiplication and infects neighboring cells. During this process an effective immune response could limit the expansion of the infection.
One of the surface molecules, eventually examined for use as a vaccine, was isolated several times from cDNA expression libraries of E. acervulina. It was originally identified by Jenkins et al. (1988, Exp. Parasitol. 66: 96-107) as a low-abundance surface protein (clone cSZ-1) detected by rabbit antibodies made to acervulina membranes. Sera from infected chickens, however did not react with it. They identified two bands in Western blots at 240 and 160 kD and showed that the β-galactosidase fusion protein, made in E. coli, could activate T-cell division. Another group (Laurent et al., (1994), Mol. Bioch. Parasitol. 63: 79-86) subsequently reported that a 19 kD surface protein, referred to as IP, was conserved across three poultry Eimeria species, shared the same cDNA sequence as the cSZ-1 clone of Jenkins et al., and suggested that iodination may have given the anomalously high molecular weights in the previous work. The nucleotide sequence of their clone was submitted to GenBank under the name of ‘19 kD sporozoite antigen’ (Accession Z26584). Most recently, Lillehoj et al. (2000, Avian Diseases 44: 379-389) isolated a slightly longer protein reading frame from a cDNA clone (3-1E) that reacted with rabbit antiserum to E. acervulina merozoites (Accession Z26584). The translation included 17 additional amino acids at the N terminus, but the remainder of the molecule was virtually identical. They demonstrated that the 3-1 E protein activated cell mediated immunity by showing in vitro IFNγ production from spleen cell isolated from chickens doubly challenged with E. acervulina and then exposed to the protein. They also showed that the protein can be used as a modestly effective vaccine to prevent coccidiosis from Eimeria. This group continues to explore this potential using the nucleotide sequence as a DNA vaccine (Song, et al., (2001), Vaccine 19: 234-252; Min, et al., (2001), Vaccine 20: 267-274). As the sole agent, the DNA vaccine generated only partial protection from challenge. Addition of plasmids carrying various chicken cytokine genes offered some improvement, but still did not reduce oocyst production by more than two-fold.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.